This proposal is aimed at understanding fundamental aspects of membrane fusion in neurons. Membrane fusion is mediated by SNARE proteins (v-SNAREs on the vesicle membrane, t-SNAREs on the target membrane) and is tightly controlled by regulatory factors. We have largely focused on the Ca2+triggered exocytosis of synaptic vesicles, which is controlled by the Ca2+binding protein synaptotagmin (syt) 1. Syt I operates through direct physical interactions with t-SNAREs and membranes. This proposal continues our studies of syt I but also extends our work to address the functions of the other fifteen isoforms of this protein. Aim 1a employs a defined reconstituted system to test the hypothesis that the syt gene family has diverged in ways that help confer specificity to intracellular membrane fusion reactions. We hypothesize that specificity is achieved, in part, through selective pairing between different isoforms of syt and t-SNAREs in various sub-cellular compartments. We will determine the syt-SNARE pairing-code and compare this with the distribution of these proteins in neurons. In Aim 1 b we continue to pursue our goal of reconstituting rapid membrane fusion that recapitulates the rapid kinetics of synaptic vesicle exocytosis in neurons. This is a key step toward defining the precise molecular mechanism that underlies exocytosis. In Aim 2 we will bridge the gap between the minimal fusion assay used in Aim 1 and studies of synaptic transmission (e.g. Aim 3 below), by analyzing directly the fusion activity of synaptic vesicles isolated from knock-out/knock-in mice. This approach compliments electrophysiological analysis because it reports the intrinsic fusion properties of vesicles lacking specific proteins. These experiments will also make it possible to address the function of synaptic vesicle proteins that we have been unable to reconstitute in an active form in Aim 1. In Aim 3, we will test the hypothesis that different isoforms of syt have diverged to impart synapses with distinct kinetic components of release. We predict that syt isoforms with fast kinetics mediate synchronous transmission, while syt isoforms with slower kinetics mediate asynchronous transmission. These studies will provide new insights into information flow in neuronal circuits. Together, the experiments proposed here will provide critical information regarding our understanding of membrane fusion at synapses. This will aid efforts to alter communication between neurons in disease states where synaptic transmission is impaired.